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Tuesday, October 15, 2013

Towards sterilizing immunity against Leptospira with a DNA vaccine

In my previous post, I described the failure of researchers to come up with a conventional protein-based subunit vaccine that confers sterilizing immunity against leptospirosis. What I mean by "conventional" subunit vaccine is a mixture of purified recombinant Leptospira protein with an adjuvant (either aluminum hydroxide or Freund's). Although an antibody response was detected against most proteins tested, immunization failed to prevent kidney colonization in every case, including those animals that survived challenge with lethal strains of Leptosipra. It's becoming clear that the leptospirosis vaccine field must move beyond simple formulations of protein plus adjuvant if sterilizing immunity is desired.

Several labs have explored more modern approaches to delivering leptospirosis vaccines. Odir Dellagostin's group down in Brazil tested the efficacy of DNA vaccines in protecting hamsters against leptospirosis, as described in this paper in Clinical and Vaccine Immunology. Forster and colleagues targeted the Leptospira interrogans LigA and LigB proteins, which are surface proteins that disrupt (or exploit) multiple host functions.

The investigators cloned various fragments of the lengthy ligA and ligB genes downstream of the human cytomegalovirus promoter of the commercial expression plasmid pTARGET. They mixed the plasmid DNA with aluminum hydroxide adjuvant and injected the material into the muscle of the hind leg of hamsters. The animals were given a booster with the same material 21 days later. An IgG immune response was detected against four of the the five Lig protein fragments being tested (see figure below).

Figure 2 from Forster et al., 2013. Sera were drawn before immunization and after the first and second immunizations with pTARGET-based lig plasmid DNA. Purified recombinant protein encoded by each plasmid was used as antigen in ELISAs. Left, middle, and right bar for each DNA: before immunization, after first immunization, and after second immunization, respectively.

21 days after the boost, the animals were challenged with a lethal strain of L. interrogans. The survival curves are shown below. Note that animals immunized with the vector alone (small filled circles) were all dead by day 11.

Figure 3 from Forster et al., 2013.

Among the lig gene fragments, the one expressing the "LigBrep" fragment was effective, protecting five of the eight animals in the group (62.5%) from death. LigBrep comprises amino acid residues 1 through 628 of LigB, whose total length is 1891 residues. Although the survival rate is nothing to get excited over, what distinguishes the LigBrep DNA vaccine from the conventional subunit vaccines tested in earlier studies is that the kidneys from 4 of the 5 survivors were culture negative, indicating that sterilizing immunity was achieved in 80% of the animals that survived infection.

Another notable outcome of the study was that protection was achieved even though the challenge strain and the vaccine's lig gene originated from different Leptospira serovars. One of the problems with killed whole-cell vaccines is that they only protect against Leptospira serovars present in the vaccine formulation because they target LPS, whose structure varies among different serovars. Leptospira proteins tend to be similar in amino acid sequence across different species and are therefore more attractive as vaccines. (The "killed-whole leptospires" control plotted in the graph above was generated from the challenge strain).

So how does DNA vaccination induce sterilizing immunity against Leptospira? As always, more studies are needed to explore this issue, but I will go ahead and speculate. DNA vaccines that are administered by standard injection stimulate a Th1-biased immune response. Studies with cattle have suggested that vaccines must stimulate Th1 immunity to minimize kidney colonization by Leptospira (see this study, for example). Moreover, an earlier study by Dellagostin's group demonstrated sterilizing immunity against L. interrogans in some animals immunized with a Mycobacterium bovis BCG strain that was engineered to express LipL32, the major outer membrane protein of L. interrogans. BCG also stimulates Th1 immunity.

Why would a Th1 response be necessary for sterilizing immunity against Leptospira? Th1 cytokines help steer B cells into producing an IgG isotype that is strongly recognized by Fc receptor on phagocytes. Consequently, bacteria bound by these IgG molecules are engulfed by opsonophagocytosis. During Leptospira infections, opsonophagocytosis clears spirochetes from the circulation during the antibody response, raising the possibility that opsonophagocytosis also leads to sterilizing immunity by vaccines that induce production of the "right" IgG.

Th1 cells are also necessary for cellular immunity, which enhances the killing functions of macrophages so that they can rid themselves of intracellular pathogens. Leptospira is considered to be an extracellular pathogen. Nevertheless, there may be a transient intracellular phase that is critical during infection. Although intracellular Leptospira has not been observed in vivo, L. interrogans is known to survive and replicate in cultured macrophages.

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Common Spirochete Diseases

Lyme disease is a tick-borne disease caused by several members of the Borrelia burgdorferi complex. B. burgdorferi, B. garinii, and B. afzelii account for most cases worldwide. A rash may appear at the site of the tick bite, and the patient may experience flu-like symptoms. Left untreated, the patient may suffer from neurologic, arthritic, and cardiac complications.

The syphilis agent Treponema pallidum is most commonly acquired by sexual contact. A skin lesion called a chancre appears at the site of initial contact with the spirochete. T. pallidum later spreads to other sites in the body to cause the flu-like symptoms and rash of secondary syphilis. Once secondary syphilis resolves, the spirochete may persist for years without causing problems. Later, tertiary syphilis can result in damage to vital tissues. Neurosyphilis and cardiovascular syphilis are two common forms of tertiary syphilis.

Leptospira lives in the kidneys of rodents and other reservoir hosts and is shed via urine into the environment. Humans acquire the spirochete by contact of abraded skin or mucous membranes with infectious urine or contaminated water or soil. Leptospirosis patients may initially experience flu-like symptoms. Jaundice and impaired kidney function occur in the potentially deadly form of leptospirosis called Weil's disease.